PCR or polymerase chain reaction, is a biotechnological tool to amplify a gene of interest. Simple PCR is used for the detection and amplification of the gene. There are various advanced methods of PCR, which are used for various biotechnological, diagnostics and research purposes. qPCR or quantitative PCR is also known as real-time PCR. qPCR is used to quantify the nucleic acids. The amplification of the DNA molecule can be monitored during the PCR, i.e. in real time. RT-PCR is referred to as a reverse transcription polymerase chain reaction. It utilises both processes, i.e. reverse transcription and polymerase chain reaction. It is used to detect and measure the amount of RNA.
People often confuse the terms PCR, RT PCR, and QPCR as the same. But they are not that same. However, the core principle in all three types remains the same. Before going to the differences of all the three PCRs, we should know what they are actually?
What is PCR?
Polymerase chain reaction or PCR is used to amplify or produce multiple copies of a gene or short stretch of DNA. It was invented by Kary Mullis. This technique is a very useful tool in molecular biology and biotechnology. It is used in labs to make billions of copies of DNA for research and diagnostics. There are three steps in PCR: Denaturation, Primer annealing and Extension of primers.
It requires two sets of primers complementary to both ends of the DNA template and a thermostable DNA polymerase. The polymerase cycle is repeated repeatedly to get multiple copies of the DNA segment.
The three steps of the polymerase chain reaction are:
Denaturation: In this step, the double-stranded DNA is separated. Single strands of DNA are formed.
Annealing: The reaction temperature is reduced to allow the complementary base pairing and annealing of the primer.
Extension: Taq polymerase, which is a thermostable DNA polymerase, is commonly used for this purpose. The DNA polymerase adds nucleotides to the primer and extends the complementary strand in the 5’-3’ direction.
What is qPCR?
qPCR or quantitative PCR is also referred to as real-time PCR. It gives an additional quantitative analysis of the DNA or RNA in RT-qPCR.
In qPCR or real-time PCR, as the name suggests, the amplification can be monitored as the PCR progresses. In qPCR, fluorescent labelling allows real-time data collection as a polymerase chain reaction is performed.
PCR products can be detected in real-time in qPCR by two methods:
Using non-specific fluorescent dyes helps in detecting double-stranded DNA. The dsDNA binding dye gives fluorescence signals as the DNA is amplified, and it increases after each cycle. The disadvantage of this method is that only one target can be examined at a time as it binds to all dsDNA fragments.
Using fluorescent-labelled DNA probes, which are sequence-specific. They can be detected after hybridization with its complementary sequence. As the DNA probes are target specific, many targets can be analysed simultaneously.
What is RT-PCR?
RT-PCR or reverse transcription PCR comprises two steps, the first is the reverse transcription process, and the second is the amplification of the desired DNA sequence by polymerase chain reaction or PCR. RT-PCR is used to detect the RNA in a sample. The amount of RNA can be measured by using RT-qPCR. RT-PCR combined with qPCR (RT-qPCR) is very useful in doing quantitative analysis of viral RNA and gene expression.
The steps of RT-PCR are the same as PCR, with the additional first step of reverse transcription. In RT-PCR, complementary DNA (cDNA) is produced first using reverse transcriptase (RT). The cDNA, thus formed, is then used as a template for the standard amplification process by PCR.
Key differences between PCR, RT PCR and qPCR:
The above content can be summarized based on the following point:
- PCR is a simple technique used to create multiple copies of the desired DNA fragments. RT PCR, on the other hand, is used for RNA amplification. On the contrary, qPCR is involved in the quantification of the nucleic acids in the respective sample.
- PCR and RT PCR are qualitative techniques, but qPCR is a quantitative technique.
- The initial template for PCR is double-stranded DNA, while RT PCR can only use RNA as template.Apart from these, qPCR has the ability to use both DNA and RNA.
- PCR is a technique with low sensitivity and specificity. In contrast, RT PCR and qPCR are very sensitive and specific.
- The resolution of the amplified product in PCR is ultra-low. On the other hand, the PCR products of RT PCR and qPCR have a higher level of resolution.
- Only the DNA polymerase enzyme is required for traditional PCR. But RT PCR and qPCR require both reverse transcriptase and DNA polymerase enzymes.
- PCR, like RT PCR, has no relevance to fluorescence, but qPCR is mainly based on the principle of fluorescence.
- The results of PCR and RT-PCR are obtained at the end of the reaction, but qPCR generates the results simultaneously in the form of peaks and graphs.
